DESCRIPTION (provided by applicant: Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. Pathogenic clinical strains o CMV differ from lab adapted strains in that they retain the ability to infect epithelial and endothelial cells. Based on the structure of the placenta endothelial cells could be a potentially important site for initiation of virus transfer across the placenta to the fetus. The mechanism of virus entry into epi/endothelial cells is different from the gB pathway of entry into fibroblast cels as it requires viral proteins gH/gL/UL128-131 forming an endocytic complex to enable viral cell entry. The guinea pig is the only small animal model that allows the study of congenital CMV by using species specific guinea pig CMV (GPCMV). We recently investigated the use of a replication-impaired live GPCMV vaccine that requires a complementing cell line for production of infectious virus. This disabled infectious single cycle (DISC) vaccine strategy results in a virs that can infect cells to express an array of viral antigens and induce an immune response but does not produce infectious virus. This vaccine strategy was highly immunogenic in guinea pigs producing antibody and T cell responses to important viral antigens. However, our original DISC vaccine lacked a newly identified homolog locus to human CMV UL128-131 (GP128-131). Consequently, this vaccine strategy lacks the ability to generate an immune response against a potential GPCMV endocytic complex. In this application we propose to define requirements for GPCMV entry into endothelial cells and additionally develop a 2nd generation DISC vaccine carrying the essential antigens necessary to induce an effective neutralizing immune response against viral infection of epi/endothelial cells. As part of this 2nd generation DISC vaccine strategy the virus will be further attenuated to increase immunogenicity and decrease the ability of the virus to establish latency by the knockout of the pp71 homolog. The ability of second generation DISC vaccines to protect against congenital GPCMV will be investigated and efficacy determined by comparing it with a recombinant gB vaccine strategy previously investigated in this model.